The long-term goal of this project is to define molecular mechanisms that control synapse development and homeostasis. Drosophila NMJ is a glutamatergic synapse, similar in structure and physiology to mammalian central excitatory synapses. In flies each NMJ is unique and identifiable, synapses are large and accessible for electrophysiological and optical analysis, making the Drosophila NMJ a powerful genetic system to study synapse development. In flies, the subunits that form the glutamate-gated ion channels (iGluRs) are known but the properties of the channels are poorly understood. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila NMJ receptors in heterologous systems and identify the structural elements and the auxiliary subunits important for receptor assembly, surface delivery, synaptic recruitment and function. We have recently solved this problem by accomplishing the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. Our studies have revealed that Drosophila iGluRs have unique ligand binding profiles: the NMJ iGluRs have low affinity for glutamate, and no response to AMPA, kainate or NMDA, while the KaiRID-containing receptors respond to kainate and are inhibited by NMDA. Furthermore, we have succeeded in expressing functional receptors in HEK293 cells, where the biophysical properties of these receptors can be further dissected using fast glutamate applications. The ability to examine the functional characteristics of iGluRs in heterologous systems opens up tremendous opportunities to study the modulation of iGluRs function and parse out a role for Neto and/or other auxiliary proteins in the receptor function vs. receptor assembly, surface expression, synaptic trafficking and/or stabilization. The Drosophila NMJ receptors consist of four different subunits: GluRIIC, -IID and -IIE, and either GluRIIA (in type-A receptors) or IIB (type-B). Various mechanisms regulating the extent of type-A and type-B receptors accumulation at synaptic sites have been described but the molecular mechanisms for the initial localization and clustering of receptors at synaptic sites remained unclear. We have recently discovered an obligatory auxiliary protein, Neto (Neuropillin and Tolloid-like), absolutely required for the iGluRs clustering and NMJ functionality. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Our investigations uncovered essential roles for Neto during synapse development and strongly support the notion that trafficking of both iGluR subtypes on the muscle membrane, their synaptic recruitment and stabilization, and their function are tightly regulated by Neto. Our results further suggest that the fly Neto isoforms (alpha and beta) directly engage iGluRs as well as other intracellular and extracellular proteins to selectively regulate the distribution of iGluRs subtypes, the recruitment of postsynaptic proteins, and the organization of postsynaptic structures. Since iGluRs gating properties control the distribution and trafficking of these receptors in vivo, Neto could influence the synaptic recruitment of iGluRs by simultaneously controlling multiple steps in receptor trafficking and clustering and/or receptor function. We also searched for novel proteins important for NMJ development using a synthetic lethality screen that takes advantage of the partial lethality of an allele with suboptimal levels of Neto. 50 % of neto hypomorphs die developmentally; further reduction of synaptogenic proteins in hemizygous animals should increase lethality. Using this strategy, we previously uncovered genetic interactions between neto and several BMP pathway components, which are known regulators of NMJ development. Recently, we have screened for relevant extracellular matrix components and discovered an interaction between neto and tenectin (tnc), which encodes a Mucin-type protein with RGD motifs. Our data indicate that Tnc is secreted from both motor neurons and muscles and accumulates in the synaptic cleft. Tnc selectively recruits alpha-PS2/beta-PS integrin at synaptic terminals, but not at the muscle attachment sites. This selectivity was instrumental in probing for integrins function during synapse development. We found that Tnc and integrin form cis active complexes with distinct pre- and postsynaptic functions. In muscle, Tnc/integrin regulates the size and architecture of synaptic boutons, partly through recruitment of the spectrin-based membrane skeleton. In motor neurons, Tnc/integrin controls neurotransmitter release. In recent studies we found that Tnc/integrin ensures the proper assembly and function of active zones by recruiting key players in neurotransmitter release, including the voltage-gated Ca2+ channel, Cacophony (Cac), and the active zone scaffold, Bruchpilot (Brp). Similar to the muscle, presynaptic Tnc/integrin also functions by anchoring the spectrin network. Together with our previous finding that postsynaptic Tnc/integrin recruits spectrin to modulate the structural integrity of synaptic boutons, this study identifies two spectrin-dependent integrin signaling pathways that coordinate synapse development and function. Based on these findings we propose that dynamic changes in the extracellular matrix of the synaptic cleft could be transduced via ligand-activated integrin and spectrin to coordinate changes in synapse structure and function.